New Findings in Genodermatoses

N e w Fi n d i n g s i n Genodermatoses Jonathan A. Dyer, MDa,b,* KEYWORDS  Genodermatoses  Genomic sequencing  Skin disease

KEY POINTS  New technologies are rapidly accelerating the rate of discovery in genetic skin diseases.  Identification of the causative gene is the first step toward defining potential translational therapies.  New discoveries continue, even for diseases such as epidermolysis bullosa or mendelian disorders of cornification (ichthyosis).  Somatic mosaicism for mutations in genes of the PI3-AKT pathway explain a variety of overgrowth syndromes.  Somatic mosaicism for mutations in genes of the RAS pathway leads to some forms of epidermal nevi suggesting these may be “mosaic RASopathies”.

The pace of the study of inherited skin diseases is rapidly accelerating. Massively parallel (nextgeneration) sequencing technologies have created paradigm shifts in how these conditions are investigated. The time it takes to generate nearcomplete genetic data on an individual is now measured in days to weeks and the cost is decreasing rapidly. Advancement is ongoing and evolving third-generation sequencing technologies have generated speculation of generating a full human genome sequence overnight. The list of new techniques, fueled by the maturation of massively parallel DNA sequencing and bioinformatics technologies to handle the resultant data deluge, is continually growing, including linkage analysis, homozygosity mapping, casecontrol association, whole-genome genotyping, targeted resequencing, and whole-exome sequencing.1 New technologies bring new challenges. The genetic causes of many inherited skin conditions are being rapidly elucidated,

leading to a deluge of genes and new data. Even for investigators in the field, keeping up with all of the new mutations and pathways at play is impractical. The subsequent deluge of “new genetic cause for disease X” articles being submitted to journals has led the Nature family of journals to publish new, more stringent guidelines for accepting articles reporting the genetic cause of a disease.2 However, it is critical to realize that the identification of causative genes is just the beginning of the journey to understanding the pathomechanism of an inherited skin disease. This article highlights a few of the many new discoveries in inherited skin diseases from the past year.

EPIDERMOLYSIS BULLOSA A group of skin disorders characterized by skin fragility, blistering, and erosion, epidermolysis bullosa (EB) occurs because of inherited defects in structural proteins critical to maintaining cell structure and adhesion in the epidermis and

Funding sources: None. Conflict of interest: None. a Department of Dermatology, University of Missouri, 1 Hospital Drive, Room MA111D, Columbia, MO 65212, USA; b Department of Child Health, University of Missouri, 1 Hospital Drive, Room MA111D, Columbia, MO 65212, USA * Department of Dermatology, University of Missouri, 1 Hospital Drive, Room MA111D, Columbia, MO 65212. E-mail address: [email protected] Dermatol Clin 31 (2013) 303–315 http://dx.doi.org/10.1016/j.det.2012.12.007 0733-8635/13/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved.

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Dyer dermoepidermal junction. Many of the EB subtypes exhibit great morbidity and some exhibit great mortality. There are several exciting new developments in EB. Several recent reports have added to the understanding of EB subtypes, including the great range in severity that occurs in different forms of EB. A second patient was recently reported with a mild form of EB simplex caused by mutations in dystonin (DST; OMIM 113810), the gene encoding bullous pemphigoid antigen type 1 (BPAG1). The first patient with a DST mutation, long hypothesized as a potential cause of EB, was reported in 2010; however, the patient also had mutations in NOTCH3 (OMIM 600276) leading to neurologic features that were likely not related to the cutaneous phenotype. However, given the concomitant diagnoses it was difficult for investigators to determine what phenotypic findings were typical for BPAG1 mutations. In the newly reported patient (with a different BPAG1 mutation), neurologic features were absent and AR mutations in the DST gene led to a very mild EB simplex phenotype with blistering only with warmth or trauma.3 A case of lethal congenital EB resulted from mutations in the junction plakoglobin (JUP; OMIM 173325) gene encoding plakoglobin (PLG). PLG plays a critical role in desmosomal function via interaction with desmoplakin, which serves to tether intermediate filaments to the desmosomal plaque. PLG is also present in adherens junctions. Previously, JUP mutations had been detected in Naxos syndrome (OMIM 601214), which exhibits wooly hair, palmoplantar keratoderma, and arrhythmogenic heart disease.4 The infant reported in this study exhibited severe skin fragility from birth with diffuse epidermal separation and massive transcutaneous water loss. The JUP mutation in this patient was unique and, instead of leading to a truncated plakoglobin protein as occurs in Naxos syndrome, led to complete PLG loss in the skin. Few desmosomes could be detected and those present were malformed. Because PLG plays a role in both desmosomal and adherens junction adhesion, its absence led to a complete loss of adhesion structures between keratinocytes.5 A new subtype of EB has been described in three patients with homozygous mutations in the integrin (ITG) a3 gene (OMIM 605025). ITGa6 (OMIM 147556) and ITGb4 (OMIM 147557) mutations are known to cause subtypes of junctional EB (JEB) often associated with pyloric atresia (OMIM 226730) and, although phenotypic variability exists, many of these patients exhibit severe skin disease and die during infancy. Patients with integrin a3 mutations had significant multiorgan disease, including a congenital nephrotic syndrome with

significant morbidity, and interstitial lung disease that is progressive and eventually lethal. Skin blistering was not a prominent feature in the immediate neonatal period and began after several months of age. Skin blistering worsened with time, with increasing fragility with trauma. Blistered areas healed slowly, with residual erythema but not scarring. The mucosa was free from lesions. Hairs were fine and sparse and, with time, onychodystrophy of the great toenails was noted as well as distal onycholysis of the fingernails. Respiratory distress or other pulmonary issues were typically the presenting complaint, with nephrotic syndrome and its resultant sequelae noted during laboratory evaluation for the respiratory problems. Ultrastructural studies did not correspond with any previously reported EB subtypes but showed disruption within the basement membrane zone.6 Thus, an infantile onset progressive EB phenotype should prompt investigation for internal organ involvement. A major source of mortality in patients with recessive dystrophic EB (RDEB) is metastatic squamous cell carcinoma. A newly published study investigates why cutaneous squamous cell carcinomas (cSCC) in patients with RDEB have a much higher rate of metastasis than standard cSCC or that seen in association with other inherited disorders such as xeroderma pigmentosum. The investigators hypothesized that type VII collagen deficiency created a permissive matrix environment for the growth and spread of cSCC. Increased type XII collagen, thrombospondin-1, and Wnt-5A were noted with deficiency of type VII collagen. Re-expression of normal type VII collagen decreased levels of these proteins, and reduced tumor cell invasion in culture and tumor growth in vivo.7 Although the details of these interactions still must be clarified, this suggests that, if type VII collagen can be replaced in patients with RDEB, skin fragility and blistering can be affected and, possibly, the risk of cSCC and the permissive growth environment can be improved. Revertant mosaicism, in which a secondary mutation occurs in a patient with an inherited disease that corrects or overcomes the primary inherited mutation, was considered a very rare event. Studies of non-Herlitz JEB (OMIM 226650) patients have shed greater light on the frequency of revertant mosaicism in these patients. Revertant mosaicism seems to be common in patients with generalized non-Herlitz JEB—possibly occurring in most patients. In patients with preexisting localized JEB, however, revertant lesions do not seem to be frequent. There does not seem to be a preference for which genetic repair mechanism is used. Such patients offer great promise as potential donors of their own reverted fibroblasts

New Findings in Genodermatoses or keratinocytes for application to blistering areas as a form of natural gene therapy.8 Translation of the understanding of the underlying genetic causes of inherited skin disease into therapies has always been a major goal of research. Chronic ulcers often become a problem in EB patients and treating them can be challenging. Jonkman and colleagues9 describe success using a punch grafting procedure using autologous donor skin for chronic ulcers in patients with laminin-332 deficient non-Herlitz JEB (OMIM 226650). Previous studies have described increased type VII collagen expression and skin adhesion in RDEB patients after injection with allogeneic fibroblasts; however, the mechanism of this increase was unclear.10 A recent report notes increased cytokine expression after allogeneic fibroblast injection, including heparin binding–EGF-like growth factor (HB-EGF), which upregulates the patient’s own COL7A1 expression.11 The effect of recombinant HB-EGF on chronic EB ulcers has not been reported but is an obvious area for future study. In the case of dystrophic EB, there have been several exciting developments focused on investigating and developing therapeutic approaches for the disease. Bone marrow–derived adult stem cells were known to play a role in maintenance of skin homeostasis; however, more recent work confirmed that these cells could generate and differentiate into skin cell types necessary for skin repair after wounding. This observation was taken to its translational conclusion with the report of a clinical trial of allogeneic bone marrow transplantation (BMT) in seven children with RDEB in 2010. Although the results of the successful BMTs were encouraging, with demonstration of detectable type VII collagen staining and clinical improvement in the patients, which lasted for more than 1 year after BMT, two of the seven children died due to complications from the BMT.12 Newer trials are underway that use conditioning regimens of significantly reduced intensity. Induced pluripotent stem cells (iPSCs) can be generated from skin fibroblasts and can serve as a renewable source of autologous stem cells. They may also be differentiated into keratinocytes.13 Initial methods of iPSC generation involved integration of viral transgenes that raised concerns that in vivo use could lead to tumor development. Currently, work is underway to develop methods of generating integration-free iPSCs. Another hurdle to the use of iPSCs is developing efficient and safe ways to correct the genetic defects present in patient-derived iPSCs. Efforts are underway to use customized zinc finger nucleases that are able to increase the incidence of homologous recombination at targeted mutation sites up to 10,000-fold. Alternatively, as noted

previously, patients with revertant mosaic skin lesions offer a potential source of autologouscorrected iPSCs.10 Direct replacement of type VII collagen using purified recombinant type VII collagen, topically and injected intravenously, is also under study. Previously, IV infusion of recombinant type VII collagen was reported to home to wounded skin and lead to functional anchoring fibril formation in a type VII collagen knockout mouse.14 Recently, similar results were described in human RDEB skin grafted onto athymic nude mice.15 An important caveat to these therapies is the concern for the potential development of antibodies against type VII collagen. Recent work suggests that patients with RDEB may have preexisting anti–type VII collagen antibodies, even when type VII collagen is not detectable in the skin. Although the source of these antibodies is not clear, one possibility is that many RDEB patients have subclinical patches of revertant fibroblasts or keratinocytes that make small amounts of type VII collagen that serve as a source for the antibody development (similar to the growing understanding of revertant mosaicism in non-Herlitz JEB). Currently, the clinical relevance of these antibodies is not known.

ICHTHYOSES The elucidation of the genetic causes of a variety of Mendelian disorders of cornification (MeDOC) has bolstered support for the bricks and mortar model of the epidermis.16 Although the role of each individual component detected to date is not completely understood, as other players are discovered the interconnections between the elements become clearer. Again, this is allowing the development of pathomechanism-based therapies for these patients. The genetic abnormalities underlying a form of lamellar ichthyosis found in golden retriever dogs led to the discovery of a new genetic cause of ichthyosis. The ichthyosis in the retrievers is similar to autosomal recessive congenital ichthyosis (ARCI) and occurs because of homozygous mutations in patatin-like phospholipase domain-containing protein 1 (PNPLA1; OMIM 612121). Although the specific function of PNPLA1 is unknown, it seems important for epidermal lipid barrier formation. On discovery of this mutation, the investigators screened ARCI patients without known mutations and found six patients from two families with PNPLA1 mutations. Affected patients could present as collodion babies and evolve into a mild-tomoderate nonbullous congenital ichthyosiform erythroderma. Erythema and keratosis involved the skin diffusely, including the flexures. A mild palmoplantar keratoderma was present and some

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Dyer patients had pseudosyndactyly of the second to third toes. Although acitretin therapy improved scaling, it worsened the erythema.17 The cause for a type of neuroichthyosis syndrome was identified in several Saudi families whose children who presented with a diffuse ichthyosis very similar to Sjo¨gren-Larsson syndrome (SLS; OMIM 270200). However, the associated neurologic phenotype in these patients was more severe than SLS, with seizures, mental retardation, and significant spasticity. These patients had been diagnosed as having ichthyosis, spastic quadriplegia, and mental retardation (ISQMR; OMIM 614457), or pseudo-SLS. Patients can present with a collodion membrane at birth. All of these patients were profoundly developmentally delayed from infancy. Seizures began in the first months of life and were unresponsive to anti-epileptics. Bilateral inguinal hernias were noted. The ichthyotic skin changes were similar to SLS and often acrally accentuated. In these patients, AR mutations in the elongation of very long chain fatty acid-like 4 gene (ELOVL4; OMIM 605512), which encodes a very long chain fatty acid synthase, were detected. Heterozygous mutations in ELOVL4 cause macular degeneration in humans (autosomal dominant [AD] macular dystrophy or Stargardt disease 3; OMIM 600110). However, ocular findings in these patients were difficult to assess but seemed absent to mild. Very long chain fatty acids play important roles in cell membrane structure and in cell signaling.18 Historically, the peeling skin syndromes (PSSs) have been a poorly understood group of rare AR syndromes characterized by episodic to continual superficial skin peeling. Acral and generalized variants are recognized with subdivision of the generalized forms into inflammatory and noninflammatory subtypes. New genetic approaches have recently allowed characterization and clarification of several of these syndromes. Some years ago, transglutaminase 5 (OMIM 603805) mutations were discovered to be the cause of the acral PSS variant (OMIM 609796) that can mimic mild forms of EB simplex.19 More recently, mutations in corneodesmosin (CDSN; OMIM 602593) were found to underlie the inflammatory variant of PSS (OMIM 270300) that is associated with allergies and significant atopic features.20 Most recently, a large family with noninflammatory AR PSS (PSS type A) was studied using whole genome homozygosity mapping followed by whole-exome sequencing of a single proband. Using this approach, a homozygous missense mutation in the carbohydrate sulfotransferase 8 (CHST8; OMIM 610190) gene was detected. CHST8 encodes an N-acetylgalactosamine-4-Osulfotransferase (GalNAc4-ST1) localized to the Golgi transmembrane and expressed in normal

human epidermis. Cells expressing the mutant enzyme had decreased levels of total sulfated glycosoaminoglycans and the investigators postulate that increased turnover of the mutant enzyme was responsible. Additionally, expression levels of GalNAc4-ST1 increase in the upper epidermal layers, suggesting a role in epidermal differentiation.21 An AR subtype of ichthyosis termed exfoliative ichthyosis (OMIM 607936) shares some features with the PSS. Affected individuals develop dry, scaling skin diffusely over the body with peeling on the palms and soles. Using a combination of genetic approaches, including whole-genome homozygosity mapping, candidate-gene analysis, and deep sequencing, loss of function (LOF) mutations in cystatin A (OMIM 184600), a protease inhibitor, were determined to be the genetic cause of exfoliative ichthyosis. Two homozygous mutations, one a splice-site and the other a nonsense mutation were detected. Microscopic analysis revealed skin disruption at the basal or suprabasal epidermal layers. Further investigation suggests that a cell-cell adhesion defect exists when cystatin A is missing. This parallels the understanding of protease inhibitor function in the normal desquamation of the epidermis as revealed by Netherton syndrome (NS; OMIM 256500). In NS, deficiency of the serine protease inhibitor (OMIM 605010) allows overactive and ongoing epidermal protease activity to continually degrade the epidermal barrier, resulting in the cutaneous stigmata of NS, the desquamative elements of which may have clinical similarity with exfoliative ichthyosis and other variants of PSS.22 A better understanding of the causes of these syndromes will enhance their clinical recognition and prevent misdiagnosis of affected patients. Also, given the role of the individual affected proteins in very specific sites of epidermal barrier function, detailed clinical and molecular study of patients with these individual disorders and with filaggrin deficiency may shed light on the exact mechanisms by which some epidermal barrier defects contribute to an inflammatory phenotype (atopic dermatitis; CDSN deficiency) while others do not. In another promising example of bench to bedside translational research, the skin lesions of congenital hemidysplasia, ichthyosis, and limb defects (CHILD) syndrome (OMIM 308050), an X-linked dominant (XLD) disorder improved with topical therapy. CHILD syndrome is caused by defects in a key component of the cholesterol biosynthesis pathway, the NAD(P)H steroid dehydrogenase-like protein (OMIM 300275). Patients with CHILD syndrome exhibit chronic recalcitrant inflammatory segmental skin lesions,

New Findings in Genodermatoses often hemilateral with associated ipsilateral limb defects. These lesions cause significant morbidity for patients and have not responded to standard systemic or topical therapies apart from sporadic reports of improvement with destructive or surgical interventions. Because the mammalian NAD(P)H steroid dehydrogenase-like protein defect leads to impaired cholesterol production, investigators first attempted topical application of cholesterol to the skin lesions to replace the deficiency, but there was no benefit. However, the addition of topical lovastatin to block overproduction of cholesterol precursors, in addition to topical cholesterol, led to significant improvement in one treated case with minimal side effects.23

INFLAMMATORY AND/OR IMMUNOLOGIC SKIN DISEASES Several recent studies have demonstrated the power of new genetic analysis strategies to identify genes of importance in cutaneous and systemic inflammatory pathways. One report describes two siblings of Lebanese descent from a consanguineous marriage with neonatal inflammatory skin and bowel disease (OMIM 614328) that developed skin lesions by the second day of life. These cutaneous lesions began as perioral and perianal erythema with fissuring and a generalized pustular rash that transitioned into psoriasiform erythroderma with periodic flares of erythema, scaling, and widespread pustules. Diarrhea began in the first week of life. In the neonatal period the diarrhea led to failure to thrive. It was bloody, clinically consistent with malabsorption, and worsened with flares in the cutaneous disease and with gastrointestinal infections. Compensatory mechanisms for the defect seem to exist in humans as there is some normalization of the gut phenotype with time. The children were prone to Staphylococcus aureus infections with recurrent blepharitis and otitis externa. Their hairs were short or broken and eyelashes and eyebrows were wiry and rough. Nail abnormalities were noted, including thickening with frequent paronychia due to recurrent candida and pseudomonal infections. The sister died at 12 years from parvovirus B19–associated myocarditis and the brother had asymptomatic left ventricular dilatation. Using single nucleotide polymorphism (SNP)homozygosity mapping, the investigators detected three large regions of homozygosity. Probes from all exons in these regions of the genome were included on a capture array and exons from these regions in the affected brother were sequenced. After known SNPs were removed, a total of 22 unique SNPs were detected in coding regions. Parallel assessment of the data for insertion-deletion variations

detected a new deletion (4bp) in a disintegrin and metalloproteinase 17 (ADAM17; tumor necrosis factor a [TNF-a] converting enzyme [TACE]; OMIM 603639), that segregated with the disease in an AR manner. This mutation predicted a severely truncated protein. Keratinocytes from the surviving sibling expressed ADAM10 (OMIM 602192), which cleaves some of the same substrates as ADAM17. Desmoglein 2 (DSG2; OMIM 125671) is a known direct target of both. Increased DSG2 expression was present in the patient’s skin suggesting a role for ADAM17 in regulating DSG2 availability at cell junctions. DSG2 is the predominant DSG in cardiac myocytes and mutations in DSG2 are associated with arrhythmogenic and dilated cardiomyopathies. Abnormal DSG2 expression may be related to the cardiac findings noted in these siblings. Because ADAM17 encodes a TACE, abnormal TNF-a signaling would be predicted to result from ADAM17 mutations and PBMCs from the surviving sibling showed impaired TNF-a production after stimulation. The low level of TNF-a detected may be from low-level production via ADAM10. The investigators also note that lack of TNF-a may have contributed to the fatal outcome in the sister because it is known to be cardioprotective in acute myocarditis.24 A recent report elucidates the genetic cause of an AD cold urticarial syndrome (familial cold autoinflammatory syndrome 3; OMIM 614468). Affected patients develop cold urticaria with generalized cold exposure rather than by touching cold objects. Similarly, patients with this subtype of cold urticaria had negative skin testing with ice-cube and coldwater immersion but positive skin testing using evaporative cooling and generalized cold air exposure. Many patients developed additional features, including a common variable immunodeficiency (75%); a susceptibility to infections (56%), especially sinopulmonary; and autoimmune disorders (56%), such as thyroiditis. Some patients also developed granulomatous skin disease. The cold urticaria began early in life and was chronic. Abnormalities in patients included low IgM and IgA, low natural killer (NK) cells, decreased circulating CD191 B cells, and IgA1 and IgG1 classswitched memory B cells. IgE was elevated in most patients. Antinuclear antibodies were present in 62%. Linkage analysis identified a single chromosomal region; however, whole-genome sequencing of one patient did not reveal any abnormalities. Analysis of a second family narrowed the linkage interval, allowing a candidate gene approach, which identified family-specific deletions in the PLCG2 gene (OMIM 600220), which encodes phospholipase Cg2 (PLCg2), a signaling molecule expressed in

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Dyer B cells, NK cells, and mast cells. The diseasecausing heterozygous deletions in PLCG2 occur in an autoinhibitory domain, leading to constitutive phospholipase activity and resulting in a gain of function (GOF) phenotype. Spontaneous degranulation below 20 C was noted in mast cells transfected with mutant PLCG2, a finding not present in controls. Also, these defects were temperature sensitive in B cells with enhanced signaling and cellular activation. The investigators propose the moniker PLCg2-associated antibody deficiency and immune dysregulation (PLAID) to identify this syndrome.25 The cause of various forms of chronic mucocutaneous candidiasis (CMC) syndromes and their causal interrelationships has been clarified greatly in several publications in the past year. Patients with CMC typically exhibit localized candida infections instead of systemic infection, due to defective cellular immunity. Defects in Th17-mediated immune function tend to result in fungal infections, reflecting the importance of Th17 cell function in the skin and/or mucosa. The pathway from fungal recognition to Th17 antifungal response involves several steps. Defects in these individual steps lead to various CMC syndromes. DECTIN1 (OMIM 606264) is a fungal pattern recognition receptor and fungal binding to dectin1 leads to signaling mediated by CARD9 (OMIM 607212). Defects in either can lead to CMC, semidominant (see later discussion) or AR in the case of dectin1 defects (OMIM 613108) and AR with CARD9 defects (OMIM 212050). In the case of DECTIN1, heterozygous carriers also showed increased Candida colonization and infections, though not at the level seen with CMC, suggesting a semidominant effect. Signal transducer and activator of transcription 3 (STAT3; OMIM 102582) plays a key role in Th17 cell differentiation and LOF in STAT3 impairs Th17 cell differentiation leading to AD hyperimmunoglobulin E syndrome (AD-HIES; OMIM 147060). Patients with AR-HIES (OMIM 243700) due to DOCK8 (OMIM 611432) mutations also exhibit impaired Th17 cell activity—albeit at a different point than that seen with STAT3 mutations. STAT1dependent (OMIM 600555) pathways inhibit Th17 cell production and heterozygous mutations that result in GOF in STAT1 decrease Th17 cell number and activity, leading to AD CMC (OMIM 614162).26 Interleukin 17 (IL-17) production is critical and mutations in the genes encoding this cytokine IL17F (OMIM 606496) and its receptor IL-17RA (OMIM 605461) inhibit Th17 cell effector function and lead to CMC, AD (OMIM 613956), and AR (OMIM 613953), respectively.27 The final step of antifungal immune response involves effective neutrophil function and impaired neutrophil

production or activity can result in CMC, such as that seen with severe congenital neutropenia.28 Large genome wide association studies (GWAS) have been performed for a variety of multigenic inflammatory skin disorders. Psoriasis was at the forefront of this research and loci identified in these studies are now being better characterized. The identity of one of these loci, PSORS2 (OMIM 602723), has long been sought. Recent reports describe GOF mutations in caspase recruitment domain family member 14 (CARD14; OMIM 607211) as the PSORS2 gene. The GOF mutations in CARD14 lead to enhanced NF-kB activation. Identified through analysis of rare families with strong penetrance of psoriasis through multiple generations, examination of large numbers of psoriasis patients from multiple cohorts has identified a variety of CARD14 variants and is beginning to shed light on their relation to NF-kB activation and possibly other mechanisms involved in the generation of psoriatic lesions.29,30

METABOLIC SYNDROMES An enlightening study has expanded the spectrum of genetic causes for pseudoxanthoma elasticum (PXE; OMIM 264800) and shed further light on potential pathomechanisms of PXE lesion development. Generalized arterial calcification of infancy (GACI; OMIM 208000) is a well recognized caused of vascular calcification in children (the other being PXE) and is typically caused by homozygous mutations in ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1; plasma cell membrane glycoprotein PC-1; OMIM 173335), which generates pyrophosphate, a critical physiologic inhibitor of calcification. However, homozygous ENPP1 mutations could not be detected in all GACI patients and some were only heterozygous for ENPP1 mutations. The vascular findings in GACI overlap significantly with PXE, which is caused by mutations in ATP-binding cassette, subfamily C, member 6 (ABCC6; OMIM 603234). Examination of ABCC6 in 28 GACI patients with less than 2 ENPP1 mutations revealed 14 patients without ENPP1 mutations who had ABCC6 mutations (8 biallelic, 6 monoallelic). Additionally, three patients with biallelic ENPP1 mutations developed skin lesions typical for PXE. These results suggest GACI and PXE are at opposite ends of a similar phenotypic spectrum and show that ABCC6 mutations account for a significant subset of GACI cases.31

OVERGROWTH SYNDROMES Proteus syndrome (OMIM 176920) has long been a challenging entity of great interest to investigators

New Findings in Genodermatoses because of its striking clinical findings and the hypothesis that John Merrick, the “Elephant Man,” suffered from this syndrome. Recently, mosaic activating mutations in v-akt murine thymoma viral oncogene homolog 1 (Akt1; OMIM 164730) have been detected in patients meeting the discrete clinical criteria for Proteus syndrome. Characterized by progressive overgrowth of skin, connective tissue, brain, and other tissues, Proteus syndrome had long been hypothesized to represent a lethal mutation surviving by somatic mosaicism. In another example of the power of new DNAsequencing technologies, investigators performed exome sequencing on DNA extracted from samples of abnormal and normal tissue in patients with Proteus syndrome. This detected a somatic activating mutation (c.49G->A, p.Glu17Lys) in the AKT1 oncogene. This encodes AKT1 kinase, which has been previously implicated in various cell functions such as proliferation and apoptosis. The investigators confirmed the presence of this mutation in a larger number of samples from patients with Proteus syndrome and AKT protein activation in lesional tissues. Of 29 subjects, activating mutations in AKT1 were found in 26. Thus, the previous hypothesis, that Proteus syndrome is caused by somatic mosaicism, was borne out. In this case it was due to activating AKT1 mutations. This likely explains past controversies regarding classification of these patients. Confusion was created when reports were published describing phosphatase and tensin homolog (PTEN; OMIM 601728) mutations in patients with Proteus syndrome. Other investigators in the field suggested that patients with PTEN mutations represented a separate segmental overgrowth syndrome similar to but distinct from Proteus syndrome in which PTEN mutations could not be detected. This PTENassociated syndrome has been designated segmental overgrowth, lipomatosis, arteriovenous malformation, and epidermal nevus (SOLAMEN) syndrome by some investigators, although Happle32 suggested the term type 2 segmental Cowden syndrome (T2SCS) because these patients carry a germline heterozygous PTEN mutation and their clinical findings result from somatic mosaic loss of heterozygosity (LOH) at the PTEN locus. Revealingly, LOF mutations in PTEN activate AKT1, and the direct linkage of these two proteins explains the overlapping, yet distinct, clinical findings in SOLAMEN and Proteus syndrome. Another disorder long postulated to represent a lethal mutation surviving by somatic mosaicism, congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES) syndrome, has also now been linked to the PI3KAKT pathway. This rare sporadic overgrowth

syndrome is not heritable. Affected patients exhibit asymmetric somatic hypertrophy as well as multiple organ anomalies. In a recent report, massively parallel sequencing was used to screen for somatic mosaic mutations in lesional tissues. Postzygotic activating mutations in phosphatidylinositol 3-kinase (PI3K), catalytic, alpha (OMIM 171834) were found in abnormal tissues from various embryonic origins, with mutant allele frequencies varying from 3% to 30%.33 These same mutations, which result in increased PI3K, have been described in cancer. PI3K activation leads to Akt1 stimulation via phosphoinositidedependent kinase 1 (PDK1; OMIM 605213) and this pathway is regulated by PTEN. Of interest to dermatologists are the keratinocytic epidermal nevi, which represent the epidermal overgrowth component of CLOVES. PIK3CA-activating mutations have previously been reported in isolated keratinocytic EN (see later discussion). The addition of CLOVES syndrome underscores that dysfunction of the PI3K-AKT pathway is common to several overgrowth syndromes. When limited to a single cell type, somatic mosaic lesions such as EN occur. However, if multiple cell lines are affected, a broader overgrowth syndrome may result.34 Additionally, it suggests the hypothesis that activating mutations of PDK1 could lead to similar overgrowth syndromes.

HAIR SYNDROMES The genetic causes of inherited hair and nail defects are also yielding to investigation. Long known because of their striking phenotype, which often led to their inclusion in circus sideshows and speculation that their condition represented a form of atavism, patients with congenital generalized hypertrichosis (CGH) syndromes represent a diverse group of inherited conditions, all with striking, diffuse, overgrowth of hair. Inheritance is variable, with some forms exhibiting AD inheritance and others exhibiting XLD. Recent reports have identified some forms of CGH as genomic, rather than simply genetic, disorders; that is, they result from genomic changes instead of than simple point mutations. Previous studies have identified copy-number variations (CNVs; in this case either microdeletions or microduplications) on chromosome 17q24 in patients with AD CGH terminalis with or without gingival hyperplasia (OMIM 135400). Chromosome 8 rearrangements were detected in hypertrichosis universalis congenita—Ambras type (OMIM 145701). Previously, linkage studies in two large Mexican families had identified a large region on the X chromosome linked to CGH. Using a large Chinese

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Dyer family with X-linked CGH (HTC2; OMIM 307150), recombination events allowed refinement of the disease locus to a critical region that overlapped with the areas reported from the Mexican families. Using a combination of high-resolution copynumber variation scan and targeted genomic sequencing, the investigators found an interchromosomal insertion on the X chromosome of a large piece of COL23A1 in affected members of the Chinese family. This insertion was just downstream of the SOX3 gene. On analysis of one of the Mexican families, a different, larger, insertion from another chromosomal region was identified inserted at the same palindromic site on the X chromosome. Because palindromic sequences are unstable and known to trigger genomic rearrangements, such as recurrent translocations and deletions, this palindromic X-chromosomal sequence likely mediates the interchromosomal insertions. The investigators suggested that these insertions could have introduced tissue-specific regulatory elements driving ectopic expression of SOX3 in follicles or their precursors with resultant abnormal hair patterning. Because some X-linked CGH pedigrees reported have had other syndromic associations, the investigators suggested different regulatory elements carried by different source chromosomal insertions in those families could result in the phenotypic variation.35 Cantu syndrome (OMIM 239850), also known as hypertrichotic osteochondrodysplasia, is a rare AD form of congenital hypertrichosis. Patients exhibit a triad of congenital hypertrichosis, cardiac defects, and distinctive coarse facial features. Cardiac defects include patent ductus arteriosus, cardiomegaly, cardiac hypertrophy, and pericardial effusion. Congenital macrosomia, macrocephaly, unusually deep palmoplantar creases, skeletal dysplasia, and recurrent respiratory infections are also reported. Whole exome sequencing of a patient and his parents led to the detection of heterozygous mutations in ATP-binding cassette, subfamily C, member 9 (ABCC9; OMIM 601439). Sequencing of ABCC9 in 15 additional cases of Cantu syndrome detected ABCC9 mutations in 13 of the 15. The two subjects in whom ABCC9 mutations could not be detected did not exhibit cardiac phenotypes. ABCC9 is part of an ATP-sensitive potassium channel complex and is widely expressed with variant splice forms. All mutations detected in Cantu syndrome patients affected both splice variants. These ATP-sensitive potassium channels couple the metabolic state of cells to their electrical activity. Four ABCC9 subunits are necessary to create a functional potassium channel and one defective subunit in the group will impair function of the channel, consistent with the dominant nature of

the mutations in Cantu syndrome. Clinical overlap between the clinical findings of Cantu syndrome and those occurring in patients treated with minoxidil has been highlighted. Tellingly, minoxidil is a potassium-ATP channel agonist via direct binding of ABCC9 subunits. This confirms the prediction that ABCC9 mutations in Cantu syndrome cause channel opening. Knowing this, potassium-ATP channel antagonists may be tested for efficacy in Cantu syndrome. The investigators note that sulfonylurea antagonizes potassium-ATP channels and has been used successfully in patients with neonatal diabetes due to activating mutations in ABCC8.36,37 Just as the genetic cause of hypertrichosis syndromes is becoming clearer, additional causes of hereditary hypotrichosis are being identified. Hereditary hypotrichosis is a clinically and genetically heterogeneous group of inherited hair loss disorders often subdivided into syndromic and nonsyndromic forms. Hereditary hypotrichosis simplex (HHS; OMIMs 146520; 605389), in contrast to other nonsyndromic hereditary hypotrichosis, does not exhibit any hair shaft changes. HHS is typically subdivided into scalp limited and generalized forms and both AD and AR inheritance are described. Several genetic causes have been reported. In several unrelated Chinese families with an AD form of generalized HHS, exome sequencing detected a mutation in the RPL21 gene (OMIM 603636), encoding a ribosomal protein called L21. These patients had onset of hair loss around 2 to 6 months of age with progression over time. Near total scalp hair loss eventually resulted. Remaining hairs in patients would reach normal length but were dry, brittle, and slow growing. Most other body hair types were also affected, except beard hairs. Teeth, nails, and sweating were normal. Ribosomal protein L21 is a part of the 60S ribosomal subunit and is highly conserved. Apart from their role in protein synthesis, understanding is growing of the role of ribosomal proteins in cellular processes outside the ribosome itself. How defects in this ribosomal protein lead to ADHHS is not yet clear but is a topic for future studies.38

NAIL SYNDROMES The causes of inherited nail diseases, or onychogenodermatoses, have not been widely investigated. A recent study identified two consanguineous pedigrees with isolated nail dysplasia inherited in an AR fashion (nonsyndromic congenital nail disorder 10, (claw-shaped nails); OMIM 614157). The clinical findings included claw-shaped nails, onychauxis (nail thickening), and onycholysis (nail separation).

New Findings in Genodermatoses Some patients have been misdiagnosed as having pachyonychia congenita (OMIM 167200; OMIM 167210) and hidrotic ectodermal dysplasia (OMIM 129500) is also considered in the differential diagnosis.15 A genome-wide SNP array was used to detect an overlapping homozygous region on the long arm of chromosome 8. Analysis of candidate genes from this highlighted region detected homozygous nonsense and missense mutations in FZD6 (OMIM 603409), encoding Frizzled 6, which belongs to a highly conserved membrane-bound Wnt receptor family involved in development and differentiation through a variety of pathways. Expression of the FZD6 missense mutation led to a shift in location of this receptor from the plasma membrane to lysosomes. The mutations described here seem to affect several Wnt-FZD pathways (canonical and noncanonical). Intact Wnt-FZD signaling is important for ectodermal appendage development, including nails. Disruption of this pathway causes a variety of conditions. For example, porcupine (PORCN; OMIM 300651), the gene defective in Goltz syndrome (focal dermal hypoplasia; OMIM 305600) is Wnt-signaling regulator. The FZD agonists R-spondin family (RSPO) member 4 (RSPO4; OMIM 610573) and RSPO1 (OMIM 609595) are defective in isolated anonychia congenita (OMIM 206800) and palmoplantar hyperkeratosis with true hermaphroditism (some with squamous cell carcinoma; OMIM 610644). Wnt10A (OMIM 606268) mutations cause odontoonychodermal dysplasia (OMIM 257980) and other ectodermal dysplasias such as Scho¨pf-SchulzPassarge syndrome (OMIM 224750) and selective tooth agenesis 4 (OMIM 150400). LMX1B (OMIM 602575) and MSX1 (OMIM 142983), Wnt-associated transcription factors, play a role in patterning and nail bed formation and are mutated in nail-patella syndrome (OMIM 161200) and Witkop syndrome (OMIM 189500), respectively.39,40

MOSAICISM Mosaic skin disorders have long been attractive candidates for gene discovery because the patient’s normal skin can serve as an ideal genetic control when compared with lesional tissue. With rapid DNA sequencing, the genetic causes of many mosaic skin lesions are being uncovered. The identification of activating mutations in the fibroblast growth factor receptor 3 (FGFR3; OMIM 134934) in nonsyndromic keratinocytic epidermal nevi (EN; OMIM 162900), suggested that some epidermal nevi represent mosaic activating mutations in known oncogenes.41 Although the initial reports described FGFR3 mutations in isolated keratinocytic EN, a recent study describes a unique

mutation in FGFR3 in a patient with a keratinocytic EN and associated syndromic features.42 In addition to FGFR3 mutations, subsequent reports described mutations in another known oncogene, phosphatidylinositol 3-kinase, catalytic, alpha (PIK3CA; OMIM 171834; see CLOVES syndrome above) in some nonsyndromic keratinocytic EN; however, these two defects were only responsible for a portion of EN cases.43 The most recent addition to this list of oncogenes is HRAS (OMIM 190020), specifically the p.G13R mutation and, rarely, KRAS (OMIM 190070). Together, these account for approximately 40% of epidermal nevi.44 The first report of RAS defects in a patient with an EN were HRAS mutations (p.G12S) detected in a middle-aged man with widespread EN and urothelial cell cancer.45 Recently, somatic mosaic HRAS and KRAS mutations have been reported to cause nevus sebaceous.46 This suggests that some forms of EN are essentially mosaic RASopathies. Recently, porokeratotic eccrine and ostial duct nevi (PEODDN) were associated with mosaicism for mutations in GJB2 (OMIM 121011), the gene encoding connexin 26, which is defective in several syndromes, including keratitis-ichthyosis-deafness (KID; OMIM 148210) syndrome.47 This finding is not unexpected because maternal mosaicism for GJB2 mutations has been reported in the mother of a child with KID syndrome. The mother presented with a widespread Blaschko-linear epidermal nevus that resembled a PEODDN. Parents of children with syndromes caused by GJB2 mutations should be examined for the presence of epidermal nevi because it could indicate underlying gonadal mosaicism and, thus, an increased recurrence risk of the GJB2-related condition with subsequent pregnancies.48

CANCER SYNDROMES The genetic cause of a new AD cancer syndrome was identified in a large family. Affected patients had a striking phenotype of telangiectasia, mild ectodermal dysplasia features (developmental anomalies of hair, teeth, and nails), and highly penetrant oropharyngeal cancer (cutaneous telangiectasia and cancer syndrome, familial; OMIM 614564). The candidate genomic region was mapped and a candidate gene approach identified a heterozygous missense mutation in the ataxia telangiectasia and Rad3-related gene (ATR; OMIM 601215), which plays a critical role in regulating genomic integrity. ATR coordinates and controls several mechanisms, including DNA replication origin firing, the stability of the DNA replication fork, cell cycle checkpoints, and DNA repair. The causative mutation occurs in a domain

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Dyer responsible for activating p53 and p53 activation was decreased in patient cells. Additionally LOH for the functional copy of the ATR gene was noted in cancer tissue from affected patients. AR loss of LOF mutations in ATR have been identified in Seckel syndrome (OMIM 21600), a developmental disorder not associated with malignancy.49 An AD familial keratoacanthoma syndrome (multiple self-healing epithelioma [MSSE]; Ferguson-Smith disease; OMIM 132800) resisted genetic analysis in the past. Though a specific region of chromosome 9q22.3 had been linked to the disease in 11 Scottish families, a lack of recombination events in the region limited further analysis and sequencing of positional candidate genes did not reveal causative mutations. With the increased throughput capacity of new generation sequencing technology, the investigators were able to evaluate a much larger region using exon capture and high throughput sequencing. This identified mutations in transforming growth factor beta receptor 1 (TGFBR1 or activin-receptor-like kinase 5; OMIM 190181), which was outside the previously suggested linkage region. Defects in TGF-b function are well known to lead to unrestricted cell growth. Missense mutations in TGFBR1 have also been implicated as a cause of AD Marfan syndrome (MFS)-like disorders, including Loeys-Dietz syndrome (LDS) 1A, OMIM 609192; LDS2A, OMIM 608967. Patients with these syndromes exhibit developmental anomalies in neurologic, skeletal and/or craniofacial, and (most importantly) cardiovascular systems but are not prone to cancer. Mutations in MSSE affect one of two regions of the protein; however, the net result of either is that no functional protein is produced. In cells heterozygous for the TGFBR1 mutation, the remaining wild-type allele is protective. However, tumors from MSSE patients exhibit LOH of the wild type TGFBR1 allele. This contrasts with MFS-related conditions in which TGFBR1 mutations result in increased TGF-b signaling instead of silencing it. How the tumors that arise in MSSE then self-resolve is not yet clear but is an area of active study.50

SKIN-DEVELOPMENT SYNDROMES Aplasia cutis congenita (ACC) has long been regarded as a spontaneous developmental disorder characterized by absence of skin at birth. Most commonly occurring on the scalp, it is often solitary. The cause of ACC is heterogeneous and a variety of conditions have been associated with its development. Adams-Oliver syndrome (AOS; OMIM 100300) is the association of ACC with terminal transverse limb defects. Most often sporadic, AOS is occasionally transmitted through families in an

AD and/or AR fashion. Various additional associated defects have been reported, including cutis marmorata and cardiac and/or vascular abnormalities. There is wide variability in severity of the phenotype, from relatively mild and subtle limb defects and small areas of ACC to extremely morbid or even mortal defects, even within families. GWAS was used to study two kindreds with AD-AOS followed by a candidate gene and exome analysis of linked areas from a single patient. This led investigators to heterozygous mutations in a RhoGAP family member, RhoGTPase-activating protein 31 (ARHGAP31; OMIM 610911), which is also known as Cdc42 GTPase-activating protein (CdGAP) and which acts to downregulate both Cdc42 (OMIM 116952) and Rac1 (OMIM 602048). Patients in this kindred had scalp ACC and variable upper and/or lower limb defects, including short distal phalanges, partially absent fingers and/or toes, and syndactyly of the second and third toes. The investigators examined Arhgap31 expression in mouse embryos, which occurred in the distal tip of the limb buds during late stages of embryonic development. Arhgap31 expression was also noted in the mouse heart although cardiac anomalies were not seen in any of the affected patients in the study. The expression of Arhgap31 in limb buds, cranium, and early heart parallel the defects reported in AOS. A screen of additional AOS patients did not reveal others with Arhgap31 mutations; thus, it is a rare cause of AOS. The mutations present in ARHGAP31 truncate the C-terminus and result in downregulation of active Cdc42 suggesting the effect of the mutations is a dominant gain of function of Arhgap31. The C-terminus of these proteins typically interacts with the N-terminal RhoGAP domain to shield a constitutively active catalytic site and lack of the C-terminus likely leads to continuous exposure of the active site. The outcome of constitutive Cdc42 inactivation is disruption of the actin cytoskeleton and ARHGAP31 functions to control the temporal and spatial cytoskeletal remodeling and modulation necessary for cell shape maintenance and migration. In summary, a variety of reports suggest that Cdc42 and Rac1 play important roles in skin and limb development and the regulation of their activity by Arhgap31 explains how GOF mutations in this protein lead to phenotype of AOS.51 A second report confirms the importance of Cdc42 and Rac1 in AOS. In patients with AR AOS, mutations in dedicator of cytokine kinesis 6 (DOCK6; OMIM 614194) were detected after autozygome analysis and exome sequencing identified LOF mutations. DOCK6 is a known modulator of Cdc42 and Rac1. Specifically, it functions to increase the amounts of active Cdc42 and Rac1; thus, deficiency of DOCK6 would be predicted to

New Findings in Genodermatoses lead to impaired activation of Cdc42 and Rac1, similar to that seen with the GOF mutations in ArhGAP31.52 These two studies shed light on the pathomechanism of AOS and also highlight the genetic heterogeneity of the condition. Clearly, defects in other members of this particular pathway or, perhaps, Cdc42 and Rac1 themselves could potentially lead to an AOS phenotype. Additionally, these findings generate the hypothesis that perhaps sporadic somatic mutations in these genes could lead to nonsyndromic localized ACC as a form of mosaicism. In this case, the undergrowth of the skin tissue would be the outcome of the somatic mosaicism instead of the overgrowth typical of EN. The limited expression of these genes to the specific body sites and time periods of embryogenesis that they demonstrate would explain the localized nature of ACC in patients carrying the germline mutation. An intriguing AD adermatoglyphia syndrome (OMIM 136000), also known as immigration delay disease,53 was investigated in a large family. Patients have a congenital absence of dermatoglyphs as well as reduced eccrine sweat gland number and overall sweating of the hands. The lack of dermatoglyphs led to problems for patients when fingerprints were required for identification, such as crossing international borders. Linkage and haplotype analysis was used to map the disease to a specific chromosomal region and a candidate gene approach identified SMARCAD1 (OMIM 612761) as a candidate gene. SMARCAD1 is a member of the SNF subfamily of a larger group of helicase proteins. Investigation of SMARCAD1 in the skin revealed the existence of a short, skin-specific (especially fibroblasts) isoform of SMARCAD1. Sequencing of SMARCAD1 from AD adermatoglyphia patients detected a heterozygous transversion that clearly segregated with the disease. This mutation disrupted a conserved donor splice site next to the 3 prime end of a noncoding exon present only in the skin specific short form of the gene, leading to aberrant splicing and poor short isoform RNA stability. How the skin-specific isoform functions in dermatoglyph or eccrine sweat gland development is not clear and a focus of ongoing study.54

SUMMARY Although this is a brief overview, it highlights a few of the discoveries in inherited skin diseases over the past 6 to 12 months. The rapid acceleration of data acquisition has allowed identification of many of the genetic causes of inherited skin diseases. However, the identification of a causative gene for a condition is simply the beginning. The genetic

causes of some inherited skin diseases, such as RDEB, have been known for decades, yet it is only recently that treatments based on that knowledge seem to be inching closer to reality. With the rapid acceleration in the pace of genetic discovery, identification of causative genes will occur in days or weeks instead of months and, it is to be hoped, a similar contraction will occur in the time needed for that knowledge to lead to therapeutic interventions for patients. Additionally, the elucidation of the genetic causes of similar diseases and syndromes greatly enhances our understanding of how different pathways interact and contribute to normal and abnormal skin development and function. The promise of this greater understanding is the potential for development of new therapies, including new applications for existing drugs, to add to our armamentarium to treat these diseases. An exciting example is the use of suppression therapy to treat genetic diseases resulting from mutations that produce premature stop codons. Various agents, such as aminoglycoside antibiotics, have the ability to induce readthrough of premature stop codons and much effort is now going toward development of more efficient safer pharmacologic agents to do the same. Already reports exist describing positive outcomes in cell culture models for disorders such as ataxia-telangiectasia, and have progressed through animal models into Phase II human trials for hemophilia A and B, methylmalonic acidemia and Duchenne muscular dystrophy. Most excitingly Phase III clinical trials are underway for cystic fibrosis.55 It has been nearly 60 years since the discovery of the structure of DNA. From then until now our understanding and appreciation of inherited skin diseases has advanced greatly. However, the accelerating rate of scientific advancement suggests a new stage of inquiry in which identification of disease-causing mutations, that critical first step in understanding and investigation, will occur rapidly and not impede pursuing a deeper understanding of the origins and pathomechanisms of cutaneous diseases.

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